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>

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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 atherogenesis23,24,25, were applied. As is shown in the present study, female OVX apoE-/- mice at weaning displayed an increase in atherosclerotic lesions in the aorta compared with female apoE-/- mice with intact ovarian function. In this animal model, we also compared the effect of various exogenous estrogen replacement therapies on atherosclerotic lesion size under a controlled dietary condition.
The double dorsal-lateral incision of bilateral ovariectomy presented in this article is technically easier, less time consuming, and safer compared with the middle dorsal-lateral incision or middle abdominal incision of bilateral ovariectomy in apoE-/- mice. Bilateral ovariectomy via middle abdominal incision presents a major drawback: it may cause severe abdominal cavity adhesion, which, in turn, may affect drug absorption. Recent reports show that peroral administration of low-dose 17&#946;-estradiol protects against cerebral ischemia26. Thus, we selected peroral administration via hazelnut spread in the present study. Commercial slow-release pellets are a frequently used administration mode for testing pharmacological effects in a mouse model but may cause detrimental cerebral damage27. Implants are prone to infections, especially if mice are subjected to bilateral OVX via a double dorsal-lateral incision. Although careful disinfection of the skin prior to incision is performed, infection is difficult to avoid. Water administration and oral gavage are two less-frequently used methods that have been tested. Administering the hormone via drinking water is highly beneficial due to its being extremely noninvasive because nearly no animal handling is required. However, 17&#946;-estradiol is not soluble in water without an emulsifier. Thus, we used DMSO at a concentration of less than 0.5% to facilitate its solution in drinking water. However, this approach for the long-term administration of low-dose DMSO is difficult to control and toxic to mice or humans. Mice may also drink water over the entire 24 h of surveillance, which makes actual drug consumption difficult to determine. Another disadvantage of this approach is that the individual&#39;s water intake is hard to control. The greatest disadvantage of oral gavage is that it is stressful for animals and may cause esophageal injury and affect eating behavior. In the methodology described here, a 5 day training of eating hormone-free hazelnut spread prior to the experiment was conducted. Approximately more than 95% of the mice will accept the hazelnut spread if being trained as what mentioned in the protocol section. Once fully habituated, most of the mice will consume it within seconds. Consistent with a previous study6,11, the plasma E2 levels decreased in OVX mice compared with the sham-operated counterpart at week 4 (data not shown). At week 12 after determination, we observed uterus atrophy in OVX-operated mice. However, the circulating estrogen levels were not monitored after OVX in the present study.
In the absence of detection of atherosclerotic lesions in the ascending aorta, the aortic IMT can be evaluated through measuring the distance between the lumen-intimal interface and the medial-adventitial interface. This measurement is based on a previously validated protocol in humans28. Average data from three sites that are approximately 100 &#181;m apart from each other. The cardiac workload increases after OVX in apoE-/- mice may due to compensatory hypertrophic growth of individual cardiomyocytes which may eventually lead to increase cardiac output (Table 1). Whereas, upon peroral administration of PPD for 9 weeks, the compensatory cardiac hypertrophy was attenuated with an EF% comparable to the sham counterpart. For validation of intra- or inter-observer variability, analyze coefficients of variation for atherosclerotic thickness measurements and parameters associated with cardiac function by one operator on two different occasions or by a different operator.
As presented in our study, OVX mice perorally administered with 17&#946;-estradiol or PPD via hazelnut spread tended to prevent body weight gain and reduce estrogen deficiency-associated lipid disruption, although no significant differences in final body weight were observed. Previous studies have also shown that lipid parameters variation may be too minor to explain the antiatherosclerotic effects of hormones29. The beneficial effects of estrogen are not limited to changes in lipid protein properties. Some nonlipid effects of estrogen30,31, such as inflammation, endothelial dysfunction, and hemodynamic stasis, can facilitate cardiovascular protection in human disease. Considering the results observed in the present study, the protection of exogenous estrogen from atherosclerosis development is partially independent of systemic lipid levels. In endothelial cells, PPD inhibited expressions of adhesion molecule and inflammatory mediators (data not shown). Furthermore, PPD could suppress perivascular adipose formation which is closely correlated with endothelial anti-contractility. Thus, the action of PPD and 17&#946;-estradiol was different and the underlying mechanism is to be further explored. Undoubtedly, excessive consumption of energy-rich food, such as hazelnut spread, could cause weight gain. However, the small amounts of hazelnut spread (200 mg&#183;kg-1&#183;day-1) as mentioned in the present study could only be responsible for less than 5% of the animals&#39; daily energy intake. Also, no obvious weight gain was detected by using this amount of hazelnut spread. The use of 17&#946;-estradiol and PPD was mainly for treatment of cardiovascular disease. Because, during the progression of atherosclerosis in OVX-operated aopE-/- mice, from Week 4 to Week 12, 17&#946;-estradiol or PPD was perorally administered.
One non-negligible point of the clinical use of HRT is its detrimental side effects, which include ovarian and breast cancers13,14. The phytoestrogen tested in the present study is a steroid saponin compound found in Dioscorea plants. PPD has been reported to have an inhibitory effect on some cancer cell lines20. In addition, PPD shows antiatherosclerotic properties comparable with those of 17&#946;-estradiol. The model we present here can help screen potential candidate compounds, including phytoestrogen, which exerts a minimal effect on tumor proliferation.

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;kg-1) or PPD (0.5 mg&#183;kg-1) was perorally administered in parallel via hazelnut spread during the last 9 weeks of treatment. All mice were weighed weekly. As shown in Figure 2, the effects of both exogenous estrogens (17&#946;-estradiol and PPD) on the plasma lipids and weekly body weight of apoE-/- mice after estrogen deficiency were evaluated. After 12 weeks of a high-cholesterol diet, OVX mice showed a remarkable increase in plasma TC and TG concentrations. OVX mice perorally administered with 17&#946;-estradiol or PPD via hazelnut spread exhibited significantly lower plasma TC concentrations than sham-operated mice (Figure 2A). Plasma TG levels decreased in OVX mice perorally administered with 17&#946;-estradiol but not with PPD (Figure 2B). As is shown in Figure 2C, while a tendency toward increased body weight (BW) was observed in OVX mice compared with the sham-operated mice, BW in OVX mice perorally administered with 17&#946;-estradiol or PPD via hazelnut spread after a high-cholesterol diet showed a tendency opposite that in OVX mice. However, the final body weight of mice in different groups showed no significant changes.
Cardiovascular function was evaluated by using echocardiography. As is shown in Figure 3, the maximal plaque or IMT of the ascending aorta was measured in OVX mice perorally administered with 17&#946;-estradiol or PPD via hazelnut spread. The aortic arch of high-cholesterol-diet-fed apoE-/- mice were observed by B-mode echocardiography. Representative longitudinal images of ascending aorta were captured by ultrasonographic biomicroscopy. The red arrows indicate the plaques. OVX mice exhibited increased maximal plaque or IMT of the ascending aorta compared with sham-operated mice. Following peroral administration of 17&#946;-estradiol or PPD via hazelnut spread, the maximal plaque or IMT of the ascending aorta remarkably decreased compared with that of OVX mice. (Figure 3). We also observed cardiac dysfunction in response to OVX in apoE-/- mice after a 12-week high-cholesterol-diet feeding (Table 1). Cardiac function was examined through echocardiography. In OVX mice, peroral administration of 17&#946;-estradiol or PPD via hazelnut spread could partially attenuate parameters showing cardiac dysfunction.
Next, we used en face analysis to determine aortic atherosclerotic lesions. As previously reported, after 12 weeks, a high-cholesterol diet led to atherosclerotic plaque formation on the luminal surface of the aorta. As shown in&#160;Figure 4, the average percentage of aortic lesion area relative to the entire aortic area significantly increased in OVX mice. Following peroral administration with 17&#946;-estradiol or PPD via hazelnut spread, the aortic lesion area remarkably decreased compared with that of the OVX mice counterpart. This result is consistent with the protection of 17&#946;-estradiol or PPD from development of atherosclerosis presented in Figure 3.
In conclusion, the proposed procedure, which uses bilateral ovariectomy via a double dorsal-lateral incision in apoE-/- mice, is applicable for screening noninvasive exogenous estrogen treatments of cardiovascular dysfunction after menopause. It is also especially useful for avoiding deleterious tumorigenetic capacity.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59536/59536fig1.jpg" /> 
Figure 1. Mouse treatment scheme. At weaning (age, 28 days), female apoE-/- C57BL/6J mice were anesthetized with avertin (tribromoethanol; 200 mg/kg; intraperitoneally). Mice were bilaterally ovariectomized (OVX) or sham operated through a 1 cm dorsal incision. One week after OVX, the mice were fed a high-cholesterol diet (1.25% cholesterol, 0% cholate) for 12 weeks. 17&#946;-Estradiol (0.1 mg&#183;kg-1) or PPD (0.5 mg&#183;kg-1) was perorally administered in parallel via hazelnut spread during the last 9 weeks of treatment. All mice were weighed every week. At week 12, cardiovascular function analysis was evaluated using echocardiography. After 12 weeks of a high-cholesterol diet, all mice were euthanized, and blood samples and tissues were harvested for further investigation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59536/59536fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59536/59536fig2.jpg" /> 
Figure 2. Effects of various exogenous estrogens on plasma lipids and weekly body weight in apoE-/- mice. Sham mice underwent a mock operation and received a high-cholesterol diet. OVX mice underwent bilateral ovariectomy and then randomly divided into the following groups: the OVX group, which was treated with a high-cholesterol diet; the OVX/E2 (17&#946;-estradiol) group, which received a high-cholesterol diet for 12 weeks plus 0.1 mg/kg E2 through peroral administration via hazelnut spread for the last 9 weeks of treatment; and the OVX/PPD group, which received a high-cholesterol diet for 12 weeks plus 0.5 mg/kg PPD through peroral administration via hazelnut spread for the last 9 weeks of treatment. The total cholesterol and triglyceride levels of plasma were measured via enzymatic methods (A-B). Data are expressed as the means &#177; SEM of n = 5 mice per group. Weekly body weights were measured from week -1 to week 12 (C). Data are expressed as the means &#177; SEM of n = 8 mice per group. One-way ANOVA followed by Dunnett&#39;s post hoc test was conducted for multiple comparisons. *p &#60; 0.05 compared with the sham group; #p &#60; 0.05 compared with the OVX group. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59536/59536fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59536/59536fig3.jpg" /> 
Figure 3. IMT or maximal plaque thickness measurements in apoE-/- mice. B-mode images showing the aortic arch of apoE-/- mice are presented. The longitudinal images of ascending aorta were obtained by ultrasonographic biomicroscopy. Maximal plaque or IMT of the ascending aorta (mm) was measured. The ultrasonographic images show plaque in the minor curvature of the ascending aorta; the red arrows indicate the plaques. Data are expressed as the mean &#177; SEM of n = 8 mice per group. One-way ANOVA followed by Dunnett&#39;s post hoc test was conducted for multiple comparisons. *p &#60; 0.05 compared with the sham group; #p &#60; 0.05 compared with the OVX group. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59536/59536fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59536/59536fig4.jpg" /> 
Figure 4. En face analysis of aortic atherosclerotic lesions in apoE-/- mice. The average percentage of aortic lesion area relative to the entire aortic area was quantified in all groups. Representative micrographs of the intimal lesions (en face) of the aorta are shown. Data are expressed as the mean &#177; SEM of n = 3 mice per group. One-way ANOVA followed by Dunnett&#39;s post hoc test was conducted for multiple comparisons. *p &#60; 0.05 compared with the sham group; #P &#60; 0.05 compared with the OVX group. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59536/59536fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Sham</td>
			<td>OVX</td>
			<td>OVX/E2</td>
			<td>OVX/PPD</td>
		</tr>
		<tr>
			<td>LVIDd (mm)</td>
			<td>3.72 &#177; 0.10</td>
			<td>3.74 &#177; 0. 24</td>
			<td>3.68 &#177; 0.16</td>
			<td>3.88 &#177; 0.16</td>
		</tr>
		<tr>
			<td>LVIDs (mm)</td>
			<td>2.34 &#177; 0.11</td>
			<td>2.16 &#177; 0.22</td>
			<td>2.12 &#177; 0.13</td>
			<td>2.55 &#177; 0.12#</td>
		</tr>
		<tr>
			<td>IVSd (mm)</td>
			<td>0.83 &#177; 0.09</td>
			<td>0.84 &#177; 0.07</td>
			<td>0.91 &#177; 0.05</td>
			<td>0.74 &#177; 0.06#</td>
		</tr>
		<tr>
			<td>IVSs (mm)</td>
			<td>1.24 &#177; 0.02</td>
			<td>1.35 &#177; 0.06*</td>
			<td>1.45 &#177; 0.04#</td>
			<td>1.09 &#177; 0.04#</td>
		</tr>
		<tr>
			<td>PWTd (mm)</td>
			<td>0.7&#177; 0.10</td>
			<td>0.68 &#177; 0.04</td>
			<td>0.72 &#177; 0.07</td>
			<td>0.58 &#177; 0.07#</td>
		</tr>
		<tr>
			<td>PWTs (mm)</td>
			<td>1.10 &#177; 0.12</td>
			<td>1.17 &#177; 0.08</td>
			<td>1.24 &#177; 0.04</td>
			<td>0.98 &#177; 0.08#</td>
		</tr>
		<tr>
			<td>EDV (mm3)</td>
			<td>58.89 &#177; 3.74</td>
			<td>59.88 &#177; 9.02</td>
			<td>57.39 &#177; 5.79</td>
			<td>65.11 &#177; 6.13</td>
		</tr>
		<tr>
			<td>ESV (mm3)</td>
			<td>18.86 &#177; 2.17</td>
			<td>15.75 &#177; 4.00</td>
			<td>14.85 &#177; 2.37</td>
			<td>23.45 &#177; 2.64#</td>
		</tr>
		<tr>
			<td>EF (%)</td>
			<td>67.84 &#177; 1.52</td>
			<td>73.91 &#177;3 .63*</td>
			<td>74.23 &#177; 1.50</td>
			<td>63.91 &#177; 3.61#</td>
		</tr>
		<tr>
			<td>FS (%)</td>
			<td>37.19 &#177; 1.53</td>
			<td>42.22 &#177; 1.17*</td>
			<td>42.36 &#177; 1.21</td>
			<td>34.28 &#177; 2.69#</td>
		</tr>
		<tr>
			<td colspan="5">LVIDd = LV internal diameter during diastole; LVIDs = LV internal diameter during systole; IVSd = internal ventricular septum during diastole; IVSs = internal ventricular septum during systole; PWTd = posterior wall thickness during diastole; PWTs = posterior wall thickness during systole; EF = ejection fraction; FS = fractional shortening; EDV = end-diastolic volume; ESV = end-systolic volume.</td>
		</tr>
	</tbody>
</table>
Table 1: Cardiac function evaluation by using Echocardiography. Parameters associated with cardiac function digitally from M-mode tracings was quantified in all groups.Data are expressed as the mean &#177; SEM of n = 8 mice per group. One-way ANOVA followed by Dunnett&#39;s post hoc test was conducted for multiple comparisons. *p &#60; 0.05 compared with the sham group; #p &#60; 0.05 compared with the OVX group.

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 ...

an-vivo-estrogen-deficiency-mouse-model-for-screening-exogenous

Nanyang Technological University

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

Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. Screening peptides affords an additional layer of complexity as conventional tip-based sample handling methods expose peptides to a large surface area of plasticware, providing an increased opportunity for peptide loss via adsorption. Preventing excessive exposure to plasticware reduces peptide loss via adherence to plastics and thus minimizes inaccuracies in potency prediction, and we have previously described the benefits of non-contact acoustic dispensing for in vitro high-throughput screening of peptide agonists1. Here we discuss a fully integrated automation solution for non-contact acoustic preparation of peptide serial dilutions in microtiter plates utilizing the example of screening for peptide agonists at the mouse glucagon-like peptide-1 receptor (GLP-1R). Our methods allow for high-throughput cell-based assays to screen for agonists and are easily scalable to support increased sample throughput, or to allow for increased numbers of assay plate copies (e.g., for a panel of more target cell lines).

Peptide adsorption to plasticware during traditional tip-based serial dilutions can significantly impact potency determination and confound the understanding of structure-activity relationships used for lead identification and lead optimization phases of drug discovery. Here methods for automated acoustic non-contact serial dilution of peptide samples are described.

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. ...

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

Activity-based protein profiling (ABPP) is a method for the identification of an enzyme of interest in a complex proteome through the use of a chemical probe that targets the enzyme&#39;s active sites. A reporter tag introduced into the probe allows for the detection of the labeled enzyme by in-gel fluorescence scanning, protein blot, fluorescence microscopy, or liquid chromatography-mass spectrometry. Here, we describe the preparation and use of the compound ARN14686, a click chemistry activity-based probe (CC-ABP) that selectively recognizes the enzyme N-acylethanolamine acid amidase (NAAA). NAAA is a cysteine hydrolase that promotes inflammation by deactivating endogenous peroxisome proliferator-activated receptor (PPAR)-alpha agonists such as palmitoylethanolamide (PEA) and oleoylethanolamide (OEA). NAAA is synthesized as an inactive full-length proenzyme, which is activated by autoproteolysis in the acidic pH of the lysosome. Localization studies have shown that NAAA is predominantly expressed in macrophages and other monocyte-derived cells, as well as in B-lymphocytes. We provide examples of how ARN14686 can be used to detect and quantify active NAAA ex vivo in rodent tissues by protein blot and fluorescence microscopy.

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

Here, we describe the preparation and use of an activity-based probe (ARN14686, undec-10-ynyl-N-[(3S)-2-oxoazetidin-3-yl]carbamate) that allows for the detection and quantification of the active form of the proinflammatory enzyme N-acylethanolamine acid amidase (NAAA), both in vitro and ex vivo.

Activity-based protein profiling (ABPP) is a method for the identification of an enzyme of interest in a complex proteome through the use of a chemical ...

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate neurotransmission that occurs within synapses because it is highly regulated by dynamic protein-protein interactions and phosphorylation events. Accordingly, when any method is used to study synaptic transmission, a major goal is to preserve these transient physiological modifications. Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments. In other words, the protocol describes a biochemical methodology to carry out protein enrichment from presynaptic, postsynaptic, and extrasynaptic compartments. First, synaptosomes, or synaptic terminals, are obtained from neurons that contain all synaptic compartments by means of a discontinuous sucrose gradient. Of note, the quality of this initial synaptic membrane preparation is critical. Subsequently, the isolation of the different subsynaptic compartments is achieved with light solubilization using mild detergents at differential pH conditions. This allows for separation by gradient and isopycnic centrifugations. Finally, protein enrichment at the different subsynaptic compartments (i.e., pre-, post- and extrasynaptic membrane fractions) is validated by means of immunoblot analysis using well-characterized synaptic protein markers (i.e., SNAP-25, PSD-95, and synaptophysin, respectively), thus enabling a direct assessment of the synaptic distribution of any particular neuronal protein.

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments.

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate ...

Measuring and Interpreting Oxygen Consumption Rates in Whole Fly Head Segments

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the progression of cancer, diabetes, neurodegeneration, and aging to name a few. For instance, changes in mitochondrial activity, the cell&#39;s metabolic powerhouse, have been characterized in many such diseases. Generally, the oxygen consumption rates of mitochondria were considered a reliable readout of mitochondrial activity and measurements in some of these studies were based on isolated mitochondria or cells. However, such conditions may not represent the complexity of a whole tissue. Recently, we have developed a novel method that enables the dynamic measurement of oxygen consumption rates from whole isolated fly heads. By utilizing this method, we have recorded lower oxygen consumption rates of the whole head segment in young versus aged flies. Secondly, we have discovered that lysine deacetylase inhibitors rapidly alter the oxygen consumption in the whole head. Our novel technique may therefore aid in uncovering new properties of various drugs, which may impact metabolic rates. Furthermore, our method may give a better understanding of metabolic behavior in an experimental setup that more closely resembles physiological states.

Measuring alterations in metabolic rates is central to understanding the progression of various diseases and aging. Here, we present a novel technique to measure whole head oxygen consumption that more closely resembles the physiological state and may aid in revealing novel drugs that modify mitochondrial activity.

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the ...

Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with clinical implications. Several methodologies have been reported to accomplish such screening. However, each has its own limitations (e.g., the screening of only ATP analogues, restriction to using purified kinase domains, significant costs associated with testing more than a few kinases at a time, and lack of flexibility in screening protein kinases with novel mutations). Here, a new protocol that overcomes some of these limitations and can be used for the unbiased screening of kinase inhibitors is presented. A strength of this method is its ability to compare the activity of kinase inhibitors across multiple proteins, either between different kinases or different variants of the same kinase. Self-assembled protein microarrays generated through the expression of protein kinases by a human-based in vitro transcription and translation system (IVTT) are employed. The proteins displayed on the microarray are active, allowing for measurement of the effects of kinase inhibitors. The following procedure describes the protocol steps in detail, from the microarray generation and screening to the data analysis.

A detailed protocol for the generation of self-assembled human protein microarrays for the screening of kinase inhibitors is presented.

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with ...

Screening for Thermotoga maritima Membrane-Bound Pyrophosphatase Inhibitors

Membrane-bound pyrophosphatases (mPPases) are dimeric enzymes that occur in bacteria, archaea, plants, and protist parasites. These proteins cleave pyrophosphate into two orthophosphate molecules, which is coupled with proton and/or sodium ion pumping across the membrane. Since no homologous proteins occur in animals and humans, mPPases are good candidates in the design of potential drug targets. Here we present a detailed protocol to screen for mPPase inhibitors utilizing the molybdenum blue reaction in a 96 well plate system. We use mPPase from the thermophilic bacterium Thermotoga maritima (TmPPase) as a model enzyme. This protocol is simple and inexpensive, producing a consistent and robust result. It takes only about one hour to complete the activity assay protocol from the start of the assay until the absorbance measurement. Since the blue color produced in this assay is stable for a long period of time, subsequent assay(s) can be performed immediately after the previous batch, and the absorbance can be measured later for all batches at once. The drawback of this protocol is that it is done manually and thus can be exhausting as well as require good skills of pipetting and time keeping. Furthermore, the arsenite-citrate solution used in this assay contains sodium arsenite, which is toxic and should be handled with necessary precautions.

Screening for Thermotoga maritima Membrane-Bound Pyrophosphatase Inhibitors

Here we present a screening method for membrane-bound pyrophosphatase (from Thermotoga maritima) inhibitors based on the molybdenum blue reaction in a 96 well plate format.

Membrane-bound pyrophosphatases (mPPases) are dimeric enzymes that occur in bacteria, archaea, plants, and protist parasites. These proteins cleave ...

Visualization of Estrogen Receptors in Colons of Mice with TNBS-Induced Crohn's Disease using Immunofluorescence

Crohn&#39;s disease is the most diagnosed type of inflammatory bowel disease. Chronic inflammation developing in the intestine leads to peristalsis disorder and damage of intestinal mucosa and seems to be associated with an increased risk of colon neoplastic transformation. Accumulating evidence indicates that estrogens and estrogen receptors affect not only hormone-sensitive tissues, but also other tissues not directly related to estrogens, such as the lungs or colon. Here, we describe the protocol for the successful immunofluorescence staining of estrogen receptors in colon obtained from a murine model of TNBS-induced Crohn&#39;s disease. A detailed protocol for the induction of Crohn&#39;s disease in mice and intestine preparation is provided as well as a step-by-step immunohistochemical procedure using formalin-fixed paraffin-embedded intestine sections. The described methods are not only useful for estrogen receptor detection and estrogen signaling investigation in vivo but can also be applied to for other proteins which may be involved in the development of colitis.

The protocol presents a complete validated TNBS-induced murine model of Crohn&#39;s disease and methods for visualization of estrogen receptors by immunohistochemistry using immunofluorescence of formalin-fixed colon sections embedded in paraffin.

Crohn&#39;s disease is the most diagnosed type of inflammatory bowel disease. Chronic inflammation developing in the intestine leads to peristalsis ...

Screening for Phytoestrogens using a Cell-based Estrogen Receptor  &#946; Reporter Assay

Plants are a source of food for many animals, and&#160;they can produce thousands of chemicals. Some of these compounds affect physiological processes in the vertebrates that consume them, such as endocrine function. Phytoestrogens, the most well studied endocrine-active phytochemicals, directly interact with the hypothalamo-pituitary gonadal axis of the vertebrate endocrine system. Here we present the novel use of a cell-based assay to screen plant extracts for the presence of compounds that have estrogenic biological activity. This assay uses mammalian cells engineered to highly express estrogen receptor beta (ER&#946;) and that have been transfected with a luciferase gene. Exposure to compounds with estrogenic activity results in the cells producing light. This assay is a reliable and simple way to test for biological estrogenic activity. It has several improvements over transient transfection assays, most notably, ease of use, the stability of the cells, and the sensitivity of the assay.

We have optimized a commercially available estrogen receptor &#946; reporter assay for screening human and nonhuman primate foods for estrogenic activity. We validated this assay by showing that the known estrogenic human food soy registers high, while other foods show no activity.

Plants are a source of food for many animals, and&#160;they can produce thousands of chemicals. Some of these compounds affect physiological processes in ...

Transplantation of Neonatal Mouse Cardiac Macrophages into Adult Mice

In an injured neonatal myocardium, macrophages facilitate cardiomyocyte proliferation and angiogenesis and promote heart regeneration. The present study reveals that transplantation of neonatal cardiac macrophages recruited by injury promotes adult heart regeneration after myocardial infarction with improvement of cardiac function and cardiomyocyte proliferation. The results indicate that neonatal cardiac macrophage transplantation could be a promising strategy for cardiac injury treatment. Here, we provide the technical details, including the isolation of neonatal cardiac macrophages from apical resection-injured neonatal mouse hearts, the transplantation of macrophages into myocardial-infarcted adult mice, and the estimation of heart regeneration after a macrophage graft.

We provide a protocol for neonatal cardiac macrophage separation and transplantation into an adult mouse heart, which could be a promising way to promote cardiac repair.

In an injured neonatal myocardium, macrophages facilitate cardiomyocyte proliferation and angiogenesis and promote heart regeneration. The present study ...

Nano-Differential Scanning Fluorimetry for Screening in Fragment-based Lead Discovery

Thermal shift assays (TSAs) examine how the melting temperature (Tm) of a target protein changes in response to changes in its environment (e.g., buffer composition). The utility of TSA, and specifically of nano-Differential Scanning Fluorimetry (nano-DSF), has been established over the years, both for finding conditions that help stabilize a specific protein and for looking at ligand binding by monitoring changes in the apparent Tm. This paper presents an efficient screening of the Diamond-SGC-iNEXT Poised (DSi-Poised) fragment library (768 compounds) by the use of nano-DSF, monitoring Tm to identify potential fragment binding. The prerequisites regarding protein quality and concentration for performing nano-DSF experiments are briefly outlined followed by a step-by-step protocol that uses a nano-liter robotic dispenser commonly used in structural biology laboratories for preparing the required samples in 96-well plates. The protocol describes how the reagent mixtures are transferred to the capillaries needed for nano-DSF measurements. In addition, this paper provides protocols to measure thermal denaturation (monitoring intrinsic tryptophan fluorescence) and aggregation (monitoring light back-scattering) and the subsequent steps for data transfer and analysis. Finally, screening experiments with three different protein targets are discussed to illustrate the use of this procedure in the context of lead discovery campaigns. The overall principle of the method described can be easily transferred to other fragment libraries or adapted to other instruments.

Monitoring changes in the melting temperature of a target protein (i.e., thermal shift assay, TSA) is an efficient method for screening fragment libraries of a few hundred compounds. We present a TSA protocol implementing robotics-assisted nano-Differential Scanning Fluorimetry (nano-DSF) for monitoring intrinsic tryptophan fluorescence and light back-scattering for fragment screening.

Thermal shift assays (TSAs) examine how the melting temperature (Tm) of a target protein changes in response to changes in its environment (e.g., buffer ...

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

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Chapters in this video

Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Measuring and Interpreting Oxygen Consumption Rates in Whole Fly Head Segments

Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays

Screening for Thermotoga maritima Membrane-Bound Pyrophosphatase Inhibitors

Visualization of Estrogen Receptors in Colons of Mice with TNBS-Induced Crohn's Disease using Immunofluorescence

Screening for Phytoestrogens using a Cell-based Estrogen Receptor β Reporter Assay

Transplantation of Neonatal Mouse Cardiac Macrophages into Adult Mice

Nano-Differential Scanning Fluorimetry for Screening in Fragment-based Lead Discovery