Biochemistry
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An In Vivo Estrogen Deficiency Mouse Model for Screening Exogenous Estrogen Treatments of Cardiovascular Dysfunction After Menopause
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Summary August 13th, 2019
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.
Transcript
Laboratory models involving estrogen deficient mice presenting an arteriosclerosis pons datas are lacking. This procedure is especially helpful for screening exogenous estrogen treatments of cardiovascular dysfunction after menopause. The main advantage of this technique is that ovariectomy without double dorsolateral incision is a easier, less time-consuming method that avoids severe abdominal cavity adhesion and inflammation.
Demonstrating the procedure will be Yue-Zhang Yin, a graduate student from my laboratory. To perform a bilateral ovariectomy in APOE knockout mice, first confirm a lack of response to the pedal reflex and place the animal in the prone position on a heating pad. Apply ointment to the animal's eyes and cover the animal with a surgical drape.
Make a one-centimeter incision lateral to the midline, and one centimeter lateral to the costal ribs, and use forceps to bluntly dissect the subcutaneous tissue. Using dissecting goggles, identify the white adipose tissue in the abdominal cavity. Using micro scissors and micro forceps, make a 0.5-to one centimeter incision through the fascia, until the abdominal cavity is reached, and use the micro forceps to gently retract the white adipose tissue.
A pink, mulberry-shaped ovary, wrapped in adipose tissue, should be observed within the abdominal cavity. Use a monofilament suture to ligate the 0.5-to one-centimeter proximal vessel and uterine horn, and use the micro scissors to harvest the ovary. Return the remaining tissue to the abdominal cavity, and use individual monofilament sutures to separately close the muscle and skin layers.
Then harvest the ovary on the contralateral side of the animal, as just demonstrated, and allow the mouse to recover on a heating pad with monitoring, until full recumbency. Once a week, from the week before to 12 weeks after the surgery, measure the body weight of each sham and experimental animal on a balance. For plasma total cholesterol and triglyceride measurement, at week 12, prepare the ventral chest area with 70%ethanol, and insert a 25-gauge needle into the right ventricle.
Aspirate slowly, until one milliliter of blood flows into the syringe, and dispense the sample into a 1.5 milliliter collection tube containing ten microliters of 0.5 molar EDTA. Invert the tube several times thoroughly to mix the EDTA into the blood, and immediately place the tube on ice. Within 30 minutes of collection, centrifuge the sample for 20 minutes at 400 times G and four degrees Celsius.
Carefully collect the plasma supernatant for storage at minus 80 degrees Celsius. To isolate the aorta, at week 12, attach a 25-gauge needle to a 50 milliliter syringe filled with PBS, and insert the needle into the left ventricle of the experimental animal. Cut the right atrium to avoid high pressure from the perfusion, and perfuse the cardiac tissue at a 05 to 08 milliliters of PBS per minute rate of flow.
Absorb the perfusion fluid with lab tissues and use scissors and forceps to remove the ribs and lungs. Open the abdominal cavity to remove the internal organs, for a better view of the aorta. Grasp the heart with micro forceps to allow separation of the aorta dorsally, from the spine, until the iliac bifurcation.
Then fix the heart and aorta for 48 hours in 4%paraformaldehyde, before storing the tissue sample in saline at room temperature or at two to eight degrees Celsius for a few hours. After twelve weeks of a high-cholesterol diet, ovariectomized mice demonstrate a remarkable increase in plasma total cholesterol and triglyceride concentrations. Ovariectomized mice, per orally administered with 17-beta oestradiol, or PPD via hazelnut spread, exhibit significantly lower plasma total cholesterol concentrations than sham-operated mice.
Plasma triglyceride levels decrease in ovariectomized mice, per orally administered with 17-beta oestradiol, but not with PPD. While a tendency toward an increased body weight is observed in ovariectomized mice, compared to sham-operated mice, no significant changes in body weight are observed overall, between the four groups of animals. The maximal plaque of the ascending aorta is increased in ovariectomized mice, compared to all other groups, in conjunction with an overall decrease in cardiac function, observed in these animals.
Further, the average percentage of aortic lesion area, relative to the entire aortic area, is significantly increased in ovariectomized mice. As expected, the presence of these lesions is decreased with 17-beta oestradiol, or PPD treatment. The main risk for the ovariectomy operation is ureteral ligation, which results in a high mortality.
Also remember to cut deeply near the renal artery branches, to avoid aortic damage.
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